The polymerase chain reaction (PCR) is a chemical method of increasing by many orders of magnitude the concentration of a specific nucleic acid sequence in a test sample. The PCR process is disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202, incorporated herein by reference. In addition, U.S. Pat. No. 4,965,188, filed Jun. 17, 1987, incorporated herein by reference, describes PCR processes in which amplification is driven by a thermostable DNA polymerase.
In PCR, a test sample believed to contain one or more targeted nucleic acid sequences is combined in a total volume of usually about 20-200 .mu.l with the following reagents: an aqueous buffer, pH 8-9 at room temperature, usually also containing approximately 0.05M KCl; all four common nucleoside triphosphates (e.g., for DNA polymerase, the four common dNTPs: dATP, dTTP, dCTP, and dGTP) at concentrations of approximately 10.sup.-5 M-10.sup.-3 M; a magnesium compound, usually MgCl.sub.2, usually at a concentration of about 1 to 5 mM; a polynucleotide polymerase, preferably a thermostable DNA polymerase, most preferably the DNA polymerase I from Thermus aquaticus (Taq polymerase, subject of U.S. Pat. No. 4,889,818, incorporated herein by reference), usually at a concentration of 10.sup.-10 to 10.sup.-8 M; and single-stranded oligonucleotide primers, usually 15 to 30 nucleotides long and usually composed of deoxyribonucleotides, containing base sequences which are Watson-Crick complementary to sequences on both strands of the target nucleic acid sequence(s). Each primer usually is present at a concentration of 10.sup.-7 to 10.sup.-5 M; primers are synthesized by solid-phase methods well known in the art of nucleic acid chemistry.
In the simplest form, PCR requires two primers for each target sequence. These primers, when annealed to the opposing target strands, have their 3' ends directed toward one another's hybridization sites and separated by about 100 to 1,000 nucleotides (occasionally up to about 10,000 nucleotides). The polymerase catalyzes magnesium-dependent, template-directed extension of each primer from the 3' end of the primer, incorporating nucleoside monophosphates into the growing nucleic acid and releasing pyrophosphate.
This extension reaction continues until the polymerase reaches the 5' end of the template strand to which the extended primer was annealed, at which point the polymerase is free to bind to another primer-template duplex and catalyze extension of that primer molecule; the extension reaction also stops if the reaction mixture is heated to temperatures sufficient to separate the template from the extended primer before the enzyme has reached the 5' end of the template. After the enzyme has worked long enough to transform a large fraction of the primer-template duplexes into double-stranded nucleic acid, the latter can be denatured at high temperature, usually 90.degree. to 100.degree. C., to create two single-stranded polynucleotides, which, after cooling to a temperature where they can be annealed to new primer molecules, serve as templates for another round of enzyme-catalyzed primer extension. Because both DNA strands serve as template, each round of nucleic acid replication approximately doubles the concentration of the specific nucleic acid sequence defined at its ends by the two primer sequences. Therefore, the total concentration increase in the target nucleic acid sequence in a PCR amplification is by a factor of approximately 2.sup.n, where n is the number of completed thermal cycles between a high temperature where double-stranded DNA is denatured and a lower temperature or set of temperatures (40.degree. to 75.degree. C.) where primer-template annealing and primer extension occur.
Although one can move PCR reaction tubes manually back and forth between thermostated baths in the two temperature ranges, PCR most commonly is performed in an automated temperature-controlled machine, known as a "thermal cycler," in which a microprocessor is programmed to change the temperature of a heat-exchange block or bath containing reaction tubes back and forth among several specified temperatures for a specified number of cycles, holding at each temperature for a specified time, usually on the order of one-half to two minutes. Such a thermal cycler is commercially available from Perkin Elmer Cetus Instruments and described in U.S. patent application Ser. No. 899,061, filed Aug. 22, 1986, which is a continuation-in-part of Ser. No. 833,368, filed Feb. 25, 1986. Both applications are now abandoned and are incorporated herein by reference. The total cycle time is usually less than 10 minutes, and the total number of cycles is usually less than 40, so that a single, multi-cycle amplification, amplifying the targeted nucleic acid sequence 10.sup.5 to 10.sup.10 times, normally takes less than seven hours and often less than four hours.
The practical benefits of PCR nucleic acid amplification have been rapidly appreciated in the fields of genetics, molecular biology, cellular biology, clinical chemistry, forensic science, and analytical biochemistry, as described in the following review volumes and articles: Erlich (ed.), 1989, PCR Technology, Stockton Press (New York); Erlich et al. (eds.), 1989, Polymerase Chain Reaction, Cold Spring Harbor Press (Cold Spring Harbor, N.Y.); Innis et al., 1990, PCR Protocols, Academic Press (New York); and White et al., 1989, Trends in Genetics 5/6:185-189. PCR can replace a large fraction of molecular cloning and mutagenesis operations commonly performed in bacteria, having advantages of speed, simplicity, lower cost, and sometime increased safety. Furthermore, PCR permits the rapid and highly sensitive qualitative and even quantitative analysis of nucleic acid sequences, often with greatly increased safety because so much PCR product is made that nonisotopic detection modes suffice.
Despite rapid and broad adoption of PCR by a range of biological and chemical disciplines, PCR still possesses several practical limitations that must be overcome for full realization of the analytical and synthetic potentials of the process. Some of these limitations are discussed in turn, below.
Many amplifications yield nonspecific side products differing in size and sequence from the sequence targeted by the primers used. Sometimes nonspecificity is caused by mis-priming, where primers have been annealed to non-target sequences, also present in the nucleic acid of the test sample similar to the target sequence. Although the genomic DNA commonly contained in PCR test samples has customarily been thought to be completely double-stranded, procedures used to prepare DNA for amplification appear to render that DNA, to a large extent, single-stranded. Single-stranded DNA is especially susceptible to mis-priming if mixed with a complete set of PCR reagents at ambient or sub-ambient temperatures. Many PCR reactions also yield primer dimers or oligomers, double-stranded side products containing the sequences of several primer molecules joined end-to-end, the yield of which correlates negatively with the yield of amplified target sequence.
The high amplification factor and resulting high sensitivity of PCR renders the process especially vulnerable to back contamination, where amplified target from one reaction is accidentally transferred into a subsequent reaction using the same primers and gives a false-positive result in the later reaction.
In principle, PCR could be performed several times faster than current practice allows, being rate limited in part by the speed of temperature change during thermal cycling. Clinical diagnostic applications of PCR would especially benefit from total amplification times of 30 to 60 minutes instead of several hours.
Lower PCR costs and increased speed and precision could be obtained if the reagents could be mixed in large batches, aliquoted into the small reaction tubes (usually one-half ml total capacity containing 20 to 200 .mu.l), and stored for long periods between preparation and use without loss of amplification efficiency.
The heretofore standard PCR art has called for covering the aqueous reaction mixture with 50 to 100 .mu.l of mineral oil to prevent solvent evaporation during the several hours of heating. The mineral oil overlay introduces several practical problems: (a) mineral oil contamination of reaction mixture samples withdrawn for post-PCR analysis, often requiring extraction with hazardous water-immiscible organic solvents to avoid interference with post-PCR processing; (b) a retardation of thermal equilibration during thermal cycling (because of the significant heat capacity of the oil layer), increasing the total cycle time; and (c) occasional introduction from some batches of mineral oil of impurities which appear to inhibit PCR, necessitating rigorous quality control of mineral oil.
The present invention significantly mitigates the limitations of PCR discussed above, by several surprisingly simple modifications of PCR practice and materials. Because primer dimer and oligomer formation can occur whenever all of the PCR reagents are mixed, even at ambient and sub-ambient temperatures in the absence of thermal cycling and in the absence of target DNA, segregation of at least one reagent from the others in a way such that all reagents do not come together before the first amplification cycle can reduce primer oligomerization and, in doing so, can greatly extend the shelf-life of the incomplete reagent mixture without greatly complicating final reaction set-up. Such segregation also can minimize mis-priming during the poorly controlled interval over which PCR reagents and test sample customarily are mixed and stored at ambient or subambient temperatures before the start of thermal cycling, especially if segregated reagents and test sample are introduced into the PCR reaction tube with minimal mixing.
Several chemical properties of magnesium confer special advantage to segregating the magnesium compound from the other PCR reagents (as opposed to segregating enzyme, primers, or dNTPs) when setting up a PCR amplification. Fatty acid salts of magnesium are potentially soluble in oil, grease, or wax, yet also potentially water extractable when the organic layer is contacted with the hot aqueous reagents during PCR. That way reagent segregation and reaction tube preparation can be simplified by incorporation of the magnesium into the organic layer rather than preparation of a separate aqueous reagent which must be added. Being inorganic, magnesium salts need not be prepared and stored with special precautions against microbial contamination, a common problem with mixtures containing nucleoside triphosphates, enzyme, or primers. The phosphatases and phosphodiesterases which degrade nucleoside triphosphates and primers often are magnesium-requiring, so that storage of the biological reagents without magnesium (possibly also with a trace of chelator to bind any small amount of magnesium present) improves shelf life and resistance to contamination by enzymes or by microbes which secrete the enzymes. Segregation of any potassium salt with the magnesium compound and away from the protein and nucleic acid also improves resistance to microbial consumption of reagents, because potassium ion also is needed for cell growth.
The present invention provides an especially effective mode of reagent segregation by providing means to replace the mineral oil overlay with a layer of grease or wax, the solidity of which at room temperature or below creates a barrier against mixing of aqueous reagents segregated above and below the grease or wax layer. Thermal cycling tans the solid barrier into a lighter-than-water liquid, which is displaced by an aqueous layer above; the upper aqueous layer contains all PCR reagents not present in the lower aqueous layer. Consequently, reagents previously segregated mix to create a complete reaction with the help of the considerable thermal convection which accompanies heating of the reaction tube. The melted grease or wax creates a vapor barrier to minimize solvent evaporation during thermal cycling and, upon cooling after amplification is complete, re-forms a solid barrier which, among other things, reduces the ease of PCR product dispersal into the environment when reaction tubes are opened, thereby reducing the likelihood of back-contaminating later reactions.
U.S. patent application Ser. No. 428,494, filed Oct. 26, 1989, now U.S. Pat. No. 5,221,608, incorporated herein by reference, describes the irradiation of psoralen and isopsoralen derivatives to photo-sterilize PCR product in a way which permits post-PCR analysis but prevents use of that product as a template in subsequent amplifications. Such photo-sterilization prevents back-contamination. However, the psoralen and isopsoralen photoreagents, commonly added before amplification, appear occasionally to inhibit PCR. Furthermore, the magnesium ion required for PCR is likely to reduce the affinity of photoreagent for double-stranded nucleic acid, thereby reducing photoreagent efficiency or increasing greatly the photoreactant concentration required for practical photo-sterilization. The replacement of mineral oil with grease or wax, as provided by the present invention, permits a practical modification of the photo-sterilization procedure that prevents interference of the reagent with amplification of new target nucleic acid and should increase photoreaction efficiency. After amplification in the absence of photoreagent and after re-solidification of the grease or wax, the reaction tube can be opened without fear of contaminating the environment with PCR product. An aqueous solution of photoreagent and a chelating reagent which binds magnesium can be placed on top of the grease or wax. Closure of the tube and a simple brief heating step to melt the grease or wax allows mixing of photoreagent and chelator with PCR product; this mixture is now ready for optimal photo-sterilization, as the chelation of magnesium ion allows tight binding of photoreagent to nucleic acid.
After PCR amplification, common practice is to detect amplified nucleic acid by reacting the amplified nucleic acid with a reagent that carries an analytical signal generator or a reagent that facilitates separation of amplified nucleic acid from other components of the PCR reaction mixture. Such reagents are designed to bind very tightly to amplified nucleic acid, either because they include oligonucleotides with sequences complementary to part of the target sequence (nucleic acid probes) or because they bind to molecules, such as fluorescein and biotin, which are conveniently attached to primers or the nucleoside triphosphates incorporated into PCR product. Signal-generating substances that might be included in such detection reagents comprise radioisotopes, fluorophores, chemiluminescent moieties, electrochemiluminescent catalysts, and catalysts in general, such as enzymes. Separation-promoting substances comprise antibodies, avidin, streptavidin, biotin, high-affinity haptens like fluorescein, magnetic particles, denser-than-water particles, lattices capable of agglutination, and adsorbents capable of binding to either single-stranded or double-stranded DNA or to specific nucleic acid sequences.
Such detection reagents often are incompatible with PCR amplification, either because, like most proteins, they are inactivated by the prolonged heating in PCR or because, like most separation-promoting substances, they might inhibit PCR by removing reagents from solution. Therefore, it is generally beneficial to add PCR product detection reagents after amplification has been completed or almost completed. As in the case of photosterilization, the present invention allows such late addition to the PCR reaction tube to occur with minimal risk of contaminating the laboratory with amplified nucleic acid, because PCR product can be sealed beneath a layer of grease or wax.
Still another situation in which late addition to a PCR reaction is desirable concerns "nested primers," wherein PCR specificity is enhanced by following an initial amplification with an amplification using primers complementary to sequences not present in the original primers or primer-complementary regions but amplified by extension of the original primers. The present invention allows late addition of the internal primer pair of a nested primer system with much reduced concern about contaminating the laboratory environment with amplified nucleic acid. After such addition, only one or a few amplification cycles are needed to generate enough of the shorter PCR product to detect.
Many other situations exist in which late addition of a substance to a PCR amplification has beneficial effects on PCR sensitivity, specificity, convenience, and product analysis. In every case, the present invention advances the art by allowing that addition to occur: (a) under conditions where amplified nucleic acid is sequestered; and/or (b) at elevated temperature.